Polymeric materials that elicit controlled cell responses, and have good mechanical, optical and/or biodegradation properties, are disclosed for use in biomedical applications. Processing methods by which such polymers can be localized at a biomaterial surface are also disclosed.
Polymers currently in use for biomedical applications generally tend to be hydrophobic. As defined herein, hydrophobic refers to a material that repels water, i.e., exhibits a static contact angle with water greater than 60 degrees at 20xc2x0 C., and has a water permeability P less than 3xc3x9710xe2x88x9210 cm3(STP) cm/(cm2 s Pa). This can give rise to uncontrolled interactions between cells and adsorbed proteins at the surface of the material, which can result in a chronic inflammatory response that can lead to failure of implants and even promote tumorigenecity (Warson, The Applications of Synthetic Resin Emulsions, Benn, London (1972)). Metal or ceramic materials used in implant applications similarly can elicit undesirable cell responses.
For tissue engineering applications, it is essential that the polymeric material used to form a biodegradable scaffold for cells promote cell adhesion, migration, growth and differentiation while providing adequate structural support. Though commonly used synthetic scaffold materials such as poly(lactide), poly(glycolide), etc., and copolymers thereof, have suitable mechanical, processing and biodegradation properties, their hydrophobic nature leads to protein adsorption and denaturing on the material surface which elicits uncontrolled cell response.
The ideal surface for many biomaterials applications would resist protein adsorption while providing cells with specific chemical signals to guide adhesion, survival, growth, migration and differentiation. As used herein, the term xe2x80x9cbiomaterialxe2x80x9d refers to a nonviable material used in a medical device intended to interact with biological systems. Polymer surfaces modified with poly(ethylene oxide) have been studied in recent years for the reduction of protein adsorption at the surface of biomaterials (Paine et al. Macromolecules, 23:3104 (1990)). The objective of these surface modification schemes is the elimination of nonspecific interactions of cells with implant materials. One way in which specific chemical signals can be relayed to cells at a surface is through tethered ligands for cell surface receptors (Barret, Brit. Polym. J. 5:259 (1973)). Delivery of signals in this manner has advantages over the addition of soluble factors, as the signal is presented in a very localized manner at a controlled dose without diffusive loss (Kuhl and Griffith, Nature Medicine, 2:1002 (1996)). In addition, tethered ligands may provide more constant stimulation to cells by avoiding the down-regulation present when soluble ligands are internalized by cells. Control over spatial distribution of ligands on surfaces may also be key to guiding cell behavior. Thus systems which will allow spatial control of local ligand density, or the creation of clusters of ligands on a surface, in addition to providing control over the average surface density of ligands, are highly desirable (Kornberg et al, Proc. Natl. Acad. Sci. USA, 88:8392 (1991)).
Integrins, dimeric adhesion receptors including one of approximately ten known alpha chains paired with one of approximately six known beta chains, mediate a wide range of interactions between cells and extracellular matrix (ECM) and control cell behaviors as diverse as migration, growth, and differentiation, providing a permissive environment for the action of growth factors. For many integrins, the specificity of integrin binding to matrix proteins has been mapped to small, discrete peptide domains and new sites continue to be elucidated (Rouslahti, Ann. Rev. Cell. Dev. Biol., 12:697 (1996); Hynes, Cell, 48:549 (1987)). The prototypical example of such specificity is the RGD site first identified in fibronectin and subsequently identified in other matrix proteins. The RGD peptide enables complete replacement of adhesive finction of fibronectin for cells expressing certain integrins.
Much data supports the idea that both occupancy and clustering of integrins are required to elicit full cellular responses mediated by integrins (Clark and Brugge, Science, 268:233 (1995)). For example, full EGFR activation of MAP kinase requires integrin clustering and occupancy (Miyamoto et al, J. Cell Biol., 135:1633 (1996)). Thus, the spatial presentation of ligand in the environment, i.e., whether ligands are spaced closely enough to afford clustering of ligand-bound integrins, may influence cellular behaviors governed by integrins. Indeed, spacing of synthetic RGD ligand covalently linked to the substrate has been shown to have an influence on cell adhesion and spreading (Massia and Hubbell, J. Cell Biol., 114:1089 (1991)). At the same time, the surface concentration of an adhesion ligand such as fibronectin has been shown to have a substantial influence on integrin-mediated behaviors such as migration (DiMilla et al, J. Cell Biol., 122:729 (1993)). A recent study using self-assembled monolayers patterned in one micron adhesive/nonadhesive domains demonstrated the role of cell spreading and receptor occupancy on cell survival (Chen et al, Science, 276:1425 (1997). The length scale in that study was approximately that of a focal adhesion complex (or larger), but it is likely that clustering over much smaller length scales (3-10 integrins) is also physiologically relevant. Indeed, data suggests strongly that RGD clustering on the less than 100 nm length scale has profound effects on the integrin-mediated behavior of migration. Since both the concentration and spatial distribution of ligand influence cell response, it is desirable to have a means to vary these two parameters independently, and over a broad range of length scales (nanometers to micrometers), in order to guide cell response.
Integrins can initiate intracellular signaling cascades that overlap with those of growth factors such as epidermal growth factor (EGF). Cross-communication between adhesion and growth factor receptors may occur by direct physical association within the focal adhesions. Both types of receptors are concentrated in these structures (Miyarnoto et al, J. Cell Biol., 135:1633 (1996); Plopper et al, Mol. Biol. Cell, 6:1349 (1995)), and both receptors can stimulate some of the same down-stream effect on molecules such as MAP kinase. Close proximity of adhesion and growth factor receptors in the focal adhesion complex provides for a free flow of both positive and negative regulatory signals between the two. A number of signaling molecules have been proposed as forming this linkage; one intracellular mechanism of transmodulation is via protein kinase C (PKC)-mediated attenuation of the epidermal growth factor receptor (EGFR). It is also likely that PKC activity secondary to phospholipase Cxcex3 or phospholipase D activation by EGFR alters integrin-based substratum connections (Welsh et al, J. Cell Biol., 114:533 (1991); Ando et al, J. Cell. Physiol., 156:487 (1993)). It is thus desirable to have a method by which two or more types of signaling ligands, such as adhesion peptides and growth factors, can be simultaneously located at the surface of a biomaterial in controlled quanitity and spatial distribution.
To date, few if any model systems are able to meet both protein resistance and cell signaling surface requirements, while approaches using clinically-applicable materials have focused on hydrogels (Hern and Hubbell, J. Biomed. Mater. Res., 39:266 (1998)), which have limited physical strength and are not suitable for many applications. Other approaches for modifying the surfaces of hydrophobic polymeric materials or other biomaterials to achieve a more desirable surface composition for biomedical applications include adsorption of block copolymers, chemical grafting of polymers to the surface, and plasma deposition of an overlying film. Each of these methods suffers various disadvantages. For example, adsorbed block copolymers can be rearranged actively by cells, grafted polymers are difficult to apply at high density on a surface, and plasma deposition results in a gel-like surface structure poorly suited for controlled cell signaling. None of these methods provides a means for modifying the surface of complex three-dimensional structures such as fibrous or sponge-like tissue scaffolds, or for creating clustered ligand distributions of variable concentration and spacing on biomaterial surfaces.
It would be advantageous to provide polymer materials and processing methods that overcome the disadvantages of other biomaterials surface modification approaches. It is therefore an object of the present invention to provide polymer materials that elicit controlled cell-surface interactions by inhibiting protein adsorption, and, where appropriate, presenting controlled concentrations and spatial distributions of cell-signaling ligands on biomaterial surfaces. It is further an object of the present invention to provide processing methods by which such polymers can be placed at a biomaterial surface. It is further the object of the present invention to provide polymeric materials which can be used to create discrete nanometer- to micrometer-sized domains on a biomaterial surface that present two or more different types of ligands for regulating cellular response.
Comb-type copolymers that elicit controlled cellular response, methods by which such polymers can be localized at a surface, and methods of using such polymers for modifying the surfaces of biomedical devices are disclosed.
The polymers include a hydrophobic, water-insoluble backbone and low molecular weight, hydrophilic, non-cell binding side chains. As defined herein, non-cell binding refers to materials which exhibit no observable cell attachment after standard cell culturing assays in serum containing media for 24 hours. The molecular weight of the hydrophilic side chains is preferably above 200 Daltons and below 2000 Daltons. The backbone can be biodegradable or non-biodegradable, depending on the intended application. Biodegradable backbones are preferred for most tissue engineering, drug delivery and wound healing device applications, while non-biodegradable backbones are desirable for permanent implant, biofiltration, and cell culture plate applications A portion of the non-cell binding side chains can be end-capped with cell-signaling ligands to control the degree of cell adhesion, or other cell response, elicited by the polymer surface. In the preferred embodiment, the overall comb copolymer should have a molecular weight sufficiently high as to confer good mechanical properties to the polymer in the melt state through chain entanglements. That is, its molecular weight should be above the entanglement molecular weight, as defined by one of ordinary skill in the art. The overall molecular weight of the comb copolymer should thus be above about 10,000 Daltons, more preferably above 20,000 Daltons, and more preferably still above 30,000 Daltons.
The density of the hydrophilic side chains along the backbone of the noncopolymers depends on the length of the side chains and the water-solubility characteristics of the final polymer. The total percentage by weight of the hydrophilic side chains is between 20 and 60 percent of the total copolymer composition, preferably around 40 percent by weight. For combs incorporating hydrophilic side chains with a molecular weight of about 350 Daltons, the mole percent of segments of the backbone carrying hydrophilic side chains can be as high as 30 percent. For hydrophilic side chains with a molecular weight of about 2000 Daltons, the mole percent of segments of the backbone carrying hydrophobic side chains can be as low as 2 percent. In the preferred embodiment, the overall comb copolymer is not water-soluble. As defined herein, the term water-soluble refers to materials having a solubility in aqueous solutions of greater than 1 gram per liter. When in contact with aqueous solutions, the hydrophilic side chains swell and form a hydrated layer which repels proteins and hence resists cellular adhesion.
The non-cell binding side chains of the comb copolymer can be end-capped with cell-signaling chemical ligands in order to elicit controlled cell responses Ligands such as adhesion peptides or growth factors can be covalently or ionically attached to the ends of the side chains using known chemistries to provide specific chemical signals to cells. A defined fraction of ligand-bearing side chains can be obtained by using appropriate stoichiometric control during the coupling of the ligands to the polymers, by protecting the end-groups on those side chains which are not to be end-capped with ligands, or by combinations of these approaches. For applications where it is desirable to cluster ligands on the length scale of nanometers or tens of nanometers on a biomaterial surface, more than one ligand (on average) can be covalently attached to a single comb copolymer chain. In applications where it is desirable to incorporate two or more types of ligands in a single cluster on a biomaterial surface on the size scale of nanometers to tens of nanometers, one or more of each of the ligand types (for example, an adhesion peptide and growth factor) can be attached to a single comb copolymer chain through its side chains using known chemistries.
When adhesion peptides are coupled to the comb copolymer side chains, cells attach and spread readily on the comb copolymer surface. The amount of cell spreading and proliferation on the surface therefore can be controlled by mixing adhesion peptide-bearing comb copolymers with non-cell binding comb copolymers, for example, so that less than 20% of the combs bear an adhesion peptide. Similarly, the spatial distribution of ligand clusters on the biomaterial surface can be controlled by mixing non-cell binding comb copolymers with comb copolymers in which each chain on average has more than one ligand attached to its side chains In this case, the size of the ligand clusters (i.e., the spatial area in which the ligands are localized) is dictated by the characteristic size of the ligand-bearing comb copolymer, and can be approximated from the comb copolymer""s radius of gyration, RG, which can be calculated or experimentally determined by one of ordinary skill in the art. The comb copolymer radius of gyration can range typically between nanometers and several tens of nanometers, depending on total molecular weight, length of side chains, and environment surrounding the polymer chain, for example, other polymer chains or water molecules (P.-G. deGennes, Scaling Concepts in Polymer Physics, Cornell University Press, 1979). Thus the size of the ligand clusters, as well as the number and type of ligands per cluster, can be controlled by the synthesis conditions of the ligand-bearing comb copolymers. For example, a comb copolymer with RG==4 nm would have an area per cluster of xcfx80 RG2 or approximately 50 nm2. The number of clusters on the surface per unit surface area (on average) can be controlled by the ratio of ligand-bearing to non-cell binding combs at the surface. To achieve a surface separation distance between ligand clusters of d, where d greater than 2RG, the concentration of ligand-bearing combs should be approximately xcfx86=Vchain/(2RGd2), where Vchain is the volume occupied by a single comb copolymer chain. For example, to achieve a cluster-to-cluster distance of 20 nm with a comb copolymer which has RG=4 nm and Vchain=48 nm3, the estimated fraction of ligand-bearing combs required is 1.5 vol %. A cluster-to-cluster distance of 10 nm would require 6 vol % of the ligand-bearing comb.
Numerous methods can be used to apply the comb copolymers, or their mixtures, to various biomaterial surfaces. These methods include dip coating, spray coating, brush coating, roll coating, or spin casting a film onto the substrate, typically followed by mild heating to promote adhesion to the surface. Solid free form processes such as three dimensional printing techniques (3DP), or freeze drying methods could be used to create complex three-dimensional structures, including porous structures. In all of these processing approaches a suitable crosslinking agent might be incorporated to enhance the mechanical rigidity of the film or device.
In applications where it is desirable to use only small amounts of copolymer to modify the surface of a second, hydrophobic or non-cell regulating polymer, the comb copolymers can be added in small quantities to the second polymer and processed to achieve segregation of the comb copolymer to the surface. In preferred embodiments, the comb copolymer would comprise less than 10 wt % of the polymer mixture. Processing steps to achieve segregation include heating the mixture under a vacuum, in air, water, water vapor, CO2 or other environment which favors the comb component at the surface, at temperatures sufficiently above the glass transitions of the polymer components to provide mobility for achieving surface segregation. In the case where the second polymer component is a semicrystalline polymer, the annealing temperature should be above the glass transition but below the melting point of the polymer, to ensure that the desired shape of the device is retained. In preferred embodiments, surface segregation is achieved during a standard processing step in the manufacture of a biomedical device, such as during an extraction, autoclaving or sterilization process. In other embodiments, segregation is accomplished in an additional annealing step in a controlled environment (water, etc), after device fabrication. Such processing steps create a surface layer approximately 2RG in thickness that contains almost exclusively the comb copolymer. The observable surface properties of such annealed mixtures are substantially identical to those of the pure comb copolymers. In preferred embodiments, the comb copolymer is miscible with the second polymer to avoid phase separation in the bulk device, which might lead to poor mechanical or optical properties.
In other cases, the localization of the comb polymer to the surface of a device primarily comprised of a second, hydrophobic or non-cell regulating polymer can be accomplished during other steps of device manufacture. For example, precise placement of the comb copolymer at the surface of a device made from a second polymer can be accomplished by 3DP methods. Likewise, differences in viscosity between the comb copolymer and a second polymer when blended together can be exploited to locate the comb to the surface during melt extrusion of fibers, films or other devices. Porous or nonporous membranes, films, fibers or hollow fibers in which the comb copolymer resides at the surfaces can be prepared by phase inversion casting. In this method, a solution of the comb copolymer, the second polymer, and a mutual solvent is cast into an aqueous-based coagulation bath to form the device During the casting process, favorable interactions between the comb and the coagulation bath medium induce segregation of the comb copolymer to exterior surfaces of the film, fiber, or membrane. Cell-regulating microporous biodegradable membranes useful as temporary barrier devices in wound-healing applications can be prepared in this fashion Cell-regulating biodegradable sutures can similarly be prepared by spinning fibers from solution into an aqueous-based coagulation bath. Such surface-modified fibers can also be prepared from biodegradable or nonbiodegradable materials and fashioned into nonwoven fabric articles for biomedical applications including cell-regulating temporary barrier devices and biofiltration devices. Hollow nanoporous fibers can be prepared which have cell-regulating interior surfaces. By encapsulating cells in a portion of such a fiber, a long-term drug delivery implant could be prepared which secretes desirable products of cells in quantities regulated wholly or in part by tethered signals on the fiber inner surface. Cell-regulating biodegradable microporous scaffolds with a surface excess of comb copolymers can be prepared by freeze-drying methods by choosing a sublimating solvent which has preferential affinity to the comb copolymer component as compared to the second polymer component which forms the bulk of the device.
In all cases described above where comb copolymers are used in conjunction with a second polymer to prepare a device, the comb copolymers can be non-cell binding combs, ligand-bearing combs, or a mixture of these to achieve a desired cell response as previously described.
A further method by which the comb copolymers can be used for controlling cell response in biomedical applications is through the preparation of polymer latexes that incorporate the comb copolymers on the latex particle surfaces. Such latexes are prepared by dispersion or emulsion polymerization methods in a water-containing medium, using the comb copolymers as a stabilizing agent. The polymerization is achieved by dissolving or mixing the desired monomer, comb stabilizer and initiator in a water-containing medium. The polymer is initiated, for example, by applying heat to the solvent. The dispersion medium is a good solvent for the comb copolymer but a poor solvent for the growing polymer The hydrophobic comb backbone is chosen to be compatible with the polymer being synthesized, and thus anchors to the surface of the growing polymer particles, while the hydrophilic side chains stabilize the particles against flocculation Upon completion of the latex synthesis, the resulting latex particles are in the range of 0.1 to 10 xcexcm in size, typically dispersed at 20-70% polymer solids by weight in the dispersion medium. These systems can be employed in a variety of ways to control cell response through the comb copolymers that remain anchored to the particle surfaces.
Films or coatings can be prepared from the latex dispersions by usual methods such as dipping, brushing, rolling or casting the latex onto any surface. For coatings applied to permanent implants to control cell response, nonbiodegradable latex particles are preferred, such as acrylics. Opaque coatings may be prepared that elicit controlled cell response by employing any of the standard coating methods used to form latex films by those skilled in the art, such as those just mentioned. Alternatively, by heat-treating films at a temperature well above the glass transition of the polymer particles, the particles will coalesce into a smooth, transparent film in which the comb copolymers reside at the surface. The comb copolymers remain localized at the surface upon coalescence due either to an energetic tendency to remain at the surface, or because there is insufficient mobility for comb diffusion into the bulk of the coalesced latex film, for example, if the film is cooled below its glass transition shortly after coalescence. The latex films exhibit surface properties akin to the comb copolymers themselves, but have the advantages that only small quantities of the comb copolymer are used (typically below 1 wt % of the total latex), coatings can be easily applied from water-based suspensions, and the film-forming properties can be tailored to adhere to the substrate by judicious choice of the film-forming polymer. For example, an acrylic latex stabilized by non-cell binding comb copolymers could be used to prepare transparent acrylic coatings on acrylic intraocular lenses in order to render them resistant to cell attachment, and hence less subject to clouding over time Acrylic latexes could also be used in applications where controlled cell response is desired at the surface of permanent metal, glass or ceramic implants or other devices, including cell culture apparatus, since a high degree of adhesion is often found between oxide surfaces and acrylic polymers. For polystyrene cell culture plates or other apparatus, a cell-regulating PS latex could be used to prepare a transparent, cell-regulating coating in the manner described above.
In all cases described above where latexes are stabilized by comb copolymers, the comb copolymers might be non-cell binding combs, ligand-bearing combs, or a mixture of these to achieve a desired cell response as previously described above. Alternatively, mixed latex dispersions can be used to prepare films which contain clustered ligand regions on a surface of sizes from 0.1 to 10 micrometers. This can be achieved by mixing together dispersions of latex particles coated with non-cell binding combs and those coated with ligand-bearing combs and creating films of these mixed dispersions as described above. The size of the ligand clusters is approximately the diameter of the latex particles coated with ligand-bearing combs, while the number of clusters on the surface per unit surface area can be controlled by the ratio of ligand-bearing to non-cell binding latex particles in the mixed dispersion.
For applications where a biodegradable film is preferred, biodegradable latexes can be prepared using comb stabilizers with biodegradable backbones. Such biodegradable latexes could also be employed as drug delivery vehicles as described below.
Comb-type copolymers that elicit regulated cellular response, methods by which such polymers can be localized at a surface, and methods of using such polymers for modifying the surfaces of biomedical devices are disclosed.
These polymers are characterized by properties that are a function of the type and ratio of hydrophilic side chains to hydrophobic backbone polymers, type and number of tethered cell-signaling ligands, molecular weight, and processing conditions
I. Polymer Composition
A. Polymer Architecture
The polymers are comb-type copolymers, with a backbone formed of a hydrophobic, water-insoluble polymer and side chains formed of short, hydrophilic non-cell binding polymers, having a molecular weight of between 200 and 2000 Daltons. The hydrophobic backbone can be biodegradable or non-biodegradable, depending on the desired application. The overall comb copolymer should have a molecular weight sufficiently high in the melt state as to confer good mechanical properties to the polymer through chain entanglement, that is, its molecular weight should be above the entanglement molecular weight, as defined by one of ordinary skill in the art. The overall molecular weight of the comb copolymer should thus be above about 10,000 Daltons, more preferably above 20,000 Daltons, and more preferably still above 30,000 Daltons. The comb copolymers can be prepared by copolymerizing a hydrophilic macromonomer which contains a polymerizable chain end with a second hydrophobic monomer. Alternatively, a hydrophobic monomer can be copolymerized with a second monomer that includes suitable reactive groups through which the hydrophilic side chains can be grafted to the backbone. Alternatively, a hydrophobic monomer with a suitable reactive side group can be polymerized and a fraction of those reactive side groups can be modified by grafting hydrophilic side chains. A defined percentage of the non-cell binding side chains can be end-capped with a suitable ligand to elicit a specific cellular response.
B. Hydrophobic Polymer Backbones
1. Biodegradable Hydrophobic Polymers
Hydrophobic polymers used to impart biodegradable properties to the backbones of the comb copolymers are preferably hydrolyzable under in vivo conditions. Suitable biodegradable polymeric units include hydroxy acids or other biologically degradable polymers that yield degradation products that are non-toxic or present as normal metabolites in the body. These include poly(amino acids), poly(anhydrides), poly(orthoesters), and poly(phosphoesters). Polylactones such as poly(epsilon-caprolactone), poly(delta-valerolactone), poly(gamma-butyrolactone)and poly(beta-hydroxybutyrate), for example, are also useful. Preferred poly(hydroxy acid)s are poly(glycolic acid), poly(DL-lactic acid) and poly(L-lactic acid), or copolymers of poly(glycolic acid and poly(lactic acid). In general, these materials degrade in vivo by both non-enzymatic and enzymatic hydrolysis, and by surface or bulk erosion.
Biodegradable regions can be constructed from monomers, oligomers or polymers using linkages susceptible to biodegradation, such as ester, peptide, anhydride, orthoester, and phosphoester bonds.
2. Non-Biodegradable Hydrophobic Polymers
Representative non-biodegradable, hydrophobic polymers that could be incorporated into the backbone of the comb copolymers include polyalkylenes such as polyethylene and polypropylene, polychloroprene, polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride), polysiloxanes, polystyrene, polyurethanes and copolymers thereof, polyacrylates, such as poly(methyl (meth)acrylate), poly(ethyl (meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl (meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate) jointly referred to herein as xe2x80x9cpolyacrylatesxe2x80x9d), and copolymers and mixtures thereof The polymers include useful derivatives, including polymers having substitutions, additions of chemical groups, for example, alkyl groups, alkylene groups, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art.
Preferred non-biodegradable polymers include ethylene vinyl acetate, polyacrylates, poly(chloroprene), and copolymers and mixtures thereof
C. Non-cell Binding Hydrophilic Side Chains
The non-cell binding side chains are preferably water-soluble when not attached to the backbone, and, more preferably, are non-ionic. Suitable polymeric blocks include those prepared from poly(ethylene glycol), poly(ethylene oxide), partially or fully hydrolyzed poly(vinyl alcohol), poly(vinylpyrrolidone), and dextran. Preferably, the side chains are made from poly(ethylene glycol), poly(ethylene oxide), or poly(acrylic acids).
The hydrophilic side chains may be intrinsically biodegradable or may be poorly biodegradable or effectively non-biodegradable in the body. In the latter two cases, the side chains should be of sufficiently low molecular weight to allow excretion. The preferred molecular weight range is below about 2000 Daltons, more preferably below 1000 Daltons, and most preferably, below about 500 Daltons. When the polymer is polyethylene glycol, it is preferred that the number of ethylene oxide monomeric units is between about 4 and 20.
When double-bond containing monomers are used to prepare the polymer backbone, a preferred method for incorporating the hydrophilic side chains is to use a hydrophilic macromonomer with a reactive double bond at one end which can be randomly incorporated during free radical or other addition polymerization. An example of such a macromonomer is PEG-methacrylate. The density of the non-cell binding, hydrophilic side chains along the polymer backbone is controlled by controlling the relative amounts of the PEG-methacrylate or other suitable macromonomeric unit used.
In those embodiments in which the side groups are end capped with cell-signaling ligands, appropriate functional groups, such as xe2x80x94NH2, xe2x80x94OH, or COOH are included on the ends of the macromonomers.
D. Monomers with Reactive Functional Groups
In many of the embodiments described herein, the monomers used to form the polymer backbone include only two reactive groups, both of which are reacted in order to form the polymer. For example, lactic acid includes two reactive groups, a hydroxy group and a carboxy group. xe2x80x94OH is the preferred reactive group. Although the ends of a polylactic acid polymer include a hydroxy group and a carboxy group, there are no reactive groups along the backbone in the final polymer chain that can be used to form a comb copolymer.
Monomers which contain one or more additional reactive groups need to be incorporated into the polymer backbone, preferably in a random fashion, in order to form the comb-type copolymers when monomers that do not include these reactive groups are used to prepare the polymer backbone. Examples of these types of monomers are well known to those of skill in the art.
The requirements for a suitable reactive monomer are that it can be incorporated in the growing polymer chain by participating in the same types of chemical reactions as the growing polymer chain. For example, when lactide is being polymerized using a Lewis acid catalyst, a depsipeptide (cyclic dimer of an amino acid) can be prepared from lysine, in which the epsilon amine group is protected, for example, with a t-boc protecting group. The lysine is incorporated into the polymer, and the protecting group can be removed. The resulting amine groups are reactive with hydrophilic polymers which include leaving groups such as tosylates, tresylates, mesylates, triflates and other leaving groups well known to those of skill in the art.
Alternatively, the reactive monomer can include a leaving group that can be displaced with a nucleophilic group on a hydrophilic polymer. For example, epichlorohydrin can be used during the polymerization step. The monomer is incorporated into the polymer backbone, and the chloride group is present on the backbone for subsequent reaction with nucleophiles. An example of a suitable hydrophilic polymer containing a nucleophilic group is a PEG with a terminal amine group. PEG-NH2 can react with the chloride groups on the polymer backbone to provide a desired density of PEG-ylation on the polymer backbone. Using the chemistry described herein, along with the general knowledge of those of skill in the art, one can prepare polymer backbones which include suitable leaving groups or nucleophiles for subsequent coupling reactions with suitably functionalized hydrophilic polymers.
E. Ligands for Controlling Cell Response
A number of molecules are known to promote cell adhesion. These can be amino acids, peptides or glycoproteins. Exemplary cell-binding ligands include peptides possessing an Arginine-Glycine-Aspartic acid (RGD) amino acid sequence or a Tyrosine-Isoleucine-Serine-Arginine-Glycine (YISRG). The RGD sequence, present in proteins such as fibronectin, has been shown to be active in promoting cell adhesion and growth (Massia, S. P. and Hubbell, J. A., J. Cell. Biol., 114:1089 (1991)). Incorporation of RGD sequences at the ends of the copolymer side chains thus can enhance cell adhesion and growth. This is particularly useful when a substrate is not adhesive, for example, a polyester to which cells such as hepatocytes show poor adhesion, which is then modified with the comb copolymer to promote cellular adhesion in a controlled manner.
Biologically active molecules may also be incorporated into the copolymer to promote the adhesion and growth of a particular cell type in vivo. Many growth factors are known and can be obtained from commercial sources such as Sigma Chemical Co, St. Louis, Mo., for example, growth factors including epidermal growth factor, vascular endothelial growth factor, fibroblast growth factor, etc.
F. Relative Ratios of Comb Components
1. Ratio of Hydrophilic to Hydrophobic Units
The density of the hydrophilic side chains along the polymer backbone depends in part on the molecular weight of the side chains. The total percent of the hydrophilic units to the hydrophobic units in the comb copolymers is between 20 and 60 percent by weight, preferably around 40 percent by weight. For hydrophilic side chains with a molecular weight of about 350, the mole percent of backbone segments carrying hydrophilic side chains can be as high as about 30 percent. For hydrophilic side chains with a molecular weight of about 2000, the mole percent can be as low as about 2 percent.
The relevant consideration when determining an appropriate ratio of hydrophilic to hydrophobic units in the comb copolymers is that the overall polymer, when the hydrophilic side chains are not end-capped with cell-signaling ligands, has the defined non-cell binding properties and preferably is not water-soluble. A relatively high density of very short (MW 500 or less) hydrophilic side chains can provide the same degree of resistance to cellular adhesion as a lower density of higher molecular weight (for example, a MW between 1500 and 2000) side chains. Those of skill in the art can adjust the molecular weight and density of the polymers taking these factors into consideration.
2. Density of Tethered Ligands
The non-cell binding side chains of the comb copolymers can be end-capped with cell-signaling chemical ligands in order to elicit specific cell responses. Ligands such as adhesion peptides or growth factors can be covalently or ionically attached to the ends of the side chains using known chemistries to provide specific chemical signals to cells. A defined fraction of ligand-bearing side chains can be obtained by using appropriate stoichiometric control during the coupling of the ligands to the ends of the side chains, by protecting the end-groups on those side chains which are not to be end-capped with ligands, or by combinations of these approaches. For applications where it is desirable to cluster ligands on the length scale of nanometers or tens of nanometers on a biomaterial surface, more than one ligand (on average) can be attached to each comb copolymer chain. In applications where it is desirable to incorporate two or more types of ligands in a single cluster on a biomaterial surface on the size scale of nanometers to tens of nanometers, one or more of each of the ligand types (for example, an adhesion peptide and growth factor) can be attached to each comb copolymer chain (on average) using known chemistries. Presentation of the ligand (or ligands) at the surface can thus be tailored in terms of overall surface density by exploiting the multi-branch nature of the comb molecule, in terms of local density, by the number of ligands attached to the same comb. The ability of the polymers to control cellular adhesion or other cell function can be adjusted by controlling the density of the cell-signaling ligands presented at the surface.
II. Polymer Mixtures
A. Mixtures of Comb Copolymers
When adhesion peptides are coupled to the comb copolymer side chains, cells attach and spread readily on the comb copolymer surface. The amount of cell spreading and proliferation on the surface therefore can be controlled by mixing adhesion peptide-bearing comb copolymers with non-cell binding comb copolymers, for example, so that less than 20%, more typically less than 2%, of the combs bear an adhesion peptide. Similarly, the spatial distribution of ligand clusters on the biomaterial surface can be controlled by mixing non-cell binding comb copolymers with comb copolymers in which each chain on average has more than one ligand attached to its side chains.
The size of the ligand clusters (i.e., the spatial area in which the ligands are localized) is dictated by the characteristic size of the ligand-bearing comb copolymer, and can be approximated from the comb copolymer""s radius of gyration, RG, which can be calculated or experimentally determined by one of ordinary skill in the art. The comb copolymer radius of gyration can range typically between nanometers and several tens of nanometers, depending on total molecular weight, length of side chains, and environment surrounding the polymer chain, for example, other polymer chains or water molecules. Thus the size of the ligand clusters, as well as the number and type of ligands per cluster, can be controlled by the synthesis conditions of the ligand-bearing comb copolymers. For example, a comb copolymer of RG would have an area per cluster of xcfx80 RG2. The number of clusters on the surface per unit surface area (on average) can be controlled by the ratio of ligand-bearing to non-cell binding combs at the surface. To achieve a surface separation distance between ligand clusters of d, where d greater than 2RG, the concentration of ligand-bearing combs should be approximately xcfx86=Vchain/(2RGd2), where Vchain is the volume occupied by a single comb copolymer chain.
B. Mixtures of Comb Copolymers and Other Polymers
The copolymers described herein can be blended with other polymers that do not elicit controlled cell responses. In applications where it is desirable to use the comb copolymer to modify the surface of a second, hydrophobic or non-cell regulating polymer, the comb copolymer can be added in small quantities to the second polymer and processed to achieve comb segregation to the surface. Blends of the comb copolymers with other polymers include those containing between 1 and 99% by weight of the comb copolymers, preferably less than 20 wt % of the comb copolymers, and more preferably less than 10 wt % of the comb copolymers. Processing steps to achieve comb surface segregation include heating the mixture under vacuum, in air, water, water vapor, supercritical CO2 or other environment that favors the comb component at the surface, at temperatures sufficiently above the glass transitions of the polymer components (the matrix polymer and the comb copolymer additive) to provide mobility for achieving surface segregation. In the case where the second polymer component is a semicrystalline polymer, the annealing temperature should be above the glass transition but below the melting point of the polymer, to ensure that the desired shape of the device is retained.
In preferred embodiments, surface segregation is achieved during a standard processing step in the manufacture of a biomedical device, such as during an extraction, autoclaving or sterilization process. In other embodiments, segregation is accomplished in an additional annealing step in a controlled environment (water, etc), after device fabrication. Such processing steps create a surface layer approximately 2RG in thickness that contains almost exclusively the comb copolymer. The observable surface properties of such annealed mixtures are substantially identical to those of the pure comb copolymers. In preferred embodiments, the comb copolymer is miscible with the second polymer to avoid phase separation in the bulk device, which might lead to poor mechanical or optical properties.
In other cases, the localization of the comb polymer to the surface of a device primarily comprised of a second, hydrophobic or non-cell regulating polymer can be accomplished during other steps of device manufacture. For example, precise placement of the comb copolymer at the surface of a device made from a second polymer can be accomplished by 3DP methods Likewise, differences in viscosity between the comb copolymer and a second polymer when blended together can be exploited to locate the comb to the surface during melt extrusion of fibers, films or other devices. Porous or nonporous membranes, films, fibers or hollow fibers in which the comb copolymer resides at the surfaces can be prepared by phase inversion casting. In this method, a solution of the comb copolymer, the second polymer, and a mutual solvent is cast into an aqueous-based coagulation bath to form the device. During the casting process, favorable interactions between the comb and the coagulation bath medium induce segregation of the comb copolymer to exterior surfaces of the film, fiber, or membrane. Cell-regulating microporous biodegradable membranes useful as temporary barrier devices in wound-healing applications can be prepared in this fashion. Cell-regulating biodegradable sutures can similarly be prepared by spinning fibers from solution into an aqueous-based coagulation bath. Such surface-modified fibers can also be prepared from biodegradable or nonbiodegradable materials and fashioned into nonwoven fabric articles for biomedical applications including cell-regulating temporary barrier devices and biofiltration devices. Hollow nanoporous fibers can be prepared which have cell-regulating interior surfaces. By encapsulating cells in a portion of such a fiber, a long-term drug delivery implant could be prepared which secretes desirable products of cells in quantities regulated wholly or in part by tethered signals on the fiber inner surface. Cell-regulating biodegradable microporous scaffolds with a surface excess of comb copolymers can be prepared by freeze-drying methods by choosing a sublimating solvent which has preferential affinity to the comb copolymer component as compared to the second polymer component which forms the bulk of the device.
In all cases described above where comb copolymers are used in conjunction with a second polymer to prepare a device, the comb copolymers can be non-cell binding combs, ligand-bearing combs, or a mixture of these to achieve a desired cell response as previously described. The observable surface properties of the device are substantially identical to those of the comb copolymer or comb copolymer mixture itself.
III. Latexes Prepared with Comb Copolymers
A. Latex Synthesis
A further method by which the comb copolymers can be used for controlling cell response in biomedical applications is through the preparation of polymer latexes that incorporate the comb copolymers on the latex particle surfaces. Such latexes can be prepared by dispersion or emulsion polymerization methods in a water-containing medium, using the comb copolymers as a stabilizing agent. The polymerization is achieved by dissolving the desired monomer, comb stabilizer and initiator in a water-containing medium. The polymer is initiated, for example, by applying heat to the solvent. The dispersion medium is a good solvent for the comb copolymer but a poor solvent for the growing polymer. The hydrophobic comb backbone is chosen to be compatible with the polymer being synthesized, and thus anchors to the surface of the growing polymer particles, while the hydrophilic side chains stabilize the particles against flocculation. Upon completion of the latex synthesis, the resulting latex particles are in the range of 0.1 to 10 xcexcm in size, typically dispersed at 20-70% polymer solids by weight in the dispersion medium. These systems can be employed in a variety of ways to control cell response through the comb copolymers that remain anchored to the particle surfaces.
Polymers which might be synthesized as latex particles for non-biodegradable applications include polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride), polystyrene, and polyacrylates, such as poly(methyl (meth)acrylate), poly(ethyl (meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl (meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate), and copolymers and mixtures thereof, as well as useful derivatives of these polymers, including polymers having substitutions, additions of chemical groups, for example, alkyl groups, alkylene groups, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art.
Polymers which might be synthesized as latex particles for biodegradable applications include poly(amino acids), poly(anhydrides), poly(orthoesters), and poly(phosphoesters), polylactones such as poly(epsilon-caprolactone), poly(delta-valerolactone), poly(gamma-butyrolactone)and poly (beta-hydroxybutyrate), and poly(hydroxy acid)s such as poly(glycolic acid), poly(DL-lactic acid) and poly(L-lactic acid), or copolymers of poly(glycolic acid and poly(lactic acid).
B. Latex Films
Films or coatings can be prepared from the latex dispersions by usual methods such as dipping, brushing, rolling or casting the latex onto any surface. For coatings applied to permanent implants to control cell response, non-biodegradable latex particles prepared with non-biodegradable comb stabilizers are preferred. For applications where a biodegradable film is preferred, biodegradable latexes can be prepared using comb stabilizers with biodegradable backbones. Opaque coatings may be prepared that elicit controlled cell response by employing any of the standard coating methods used to form latex films, such as those just mentioned. Alternatively, by heat-treating films at a temperature well above the glass transition of the polymer particles, the particles will coalesce into a smooth, transparent film in which the comb copolymers reside at the surface. The comb copolymers remain localized at the surface upon coalescence due either to an energetic tendency to remain at the surface, or because there is insufficient mobility for comb diffusion into the bulk of the coalesced latex film, for example, if the film is cooled below its glass transition shortly after coalescence.
The latex films exhibit surface properties of the comb copolymers themselves, but have the advantages that only small quantities of the comb copolymer are required (typically below 1 wt % of the total latex), coatings can be easily applied from water-based suspensions, and the film-forming properties can be tailored to adhere to the substrate by judicious choice of the film-forming polymer. For example, an acrylic latex stabilized by non-cell binding comb copolymers could be used to prepare transparent acrylic coatings on acrylic intraocular lenses in order to render them resistant to cell attachment, and hence less subject to clouding over time. Acrylic latexes could also be used in applications where controlled cell response is desired at the surface of permanent metal, glass or ceramic implants or other devices, including cell culture apparatus, since a high degree of adhesion is often found between oxide surfaces and acrylic polymers. For polystyrene cell culture plates or other apparatus, a cell-regulating PS latex could be used to prepare a transparent, cell-regulating coating in the manner described above.
In all cases described above where latexes are stabilized by comb copolymers, the comb copolymers might be non-cell binding combs, ligand-bearing combs, or a mixture of these to achieve a desired cell response as previously described above. Alternatively, mixed latex dispersions can be used to prepare films that contain clustered ligand regions on a surface of sizes from 0.1 to 10 micrometers. This can be achieved by mixing together dispersions of latex particles coated with non-cell binding combs and those coated with ligand-bearing combs and creating films of these mixed dispersions as described above. The size of the ligand clusters is approximately the diameter of the latex particles coated with ligand-bearing combs, while the number of clusters on the surface per unit surface area can be controlled by the ratio of ligand-bearing to non-cell binding latex particles in the mixed dispersion.
IV. Polymer Preparation
Methods for preparing hydrophobic polymers including reactive monomeric units are known. Typical reactions are ring opening polymerization (for monomers such as lactide, glycolide, and other cyclic monomeric units), free radical polymerization (for double bond-containing monomeric units such as methyl methacrylate), and anionic or other addition polymerizations.
The monomers used to prepare the hydrophobic polymer backbone, for example, lactide, glycolide, caprolactone, and trimethylene carbonate, can be reacted with various polymerization initiators, for example, alcohols such as ethylene glycol and ethanol, water, and amines, in the presence of a suitable catalyst such as a Lewis acid, as described, for example, in Kricheldorf, H. R. in Models of Biopolymers by Ring-Opening Polymerization, Penczek, S., Ed., CRC Press, Boca Raton, 1990, Chapter 1; Kricheldorf, H. R. xcex1-Aminoacid-N-Carboxy-Anhydrides and Related Heterocycles, Springer-Verlag, Berlin, 1987; and Imanishi, Y. in Ring-Opening Polymerization, Ivin, K. J. and Saegusa, T., Eds., Elsevier, London, 1984, Volume 2, Chapter 8.
The cell-binding polymer side chains grafted onto the polymer backbone are preferably hydrophilic polymers, such as polyethylene glycol, polyethylene oxide, polyacrylic acid, dextran and mixtures thereof, which can be modified to include reactive functional groups such as amino, carboxylic acid, halo, sulfide, guanidino, imidazole and hydroxyl groups. These groups can react with various reactive groups on the polymer backbone in routine nucleophilic displacement reactions to graft the hydrophilic polymers to the backbone. The side chain polymers can be end-capped with cell binding ligands through standard covalent or ionic coupling reactions.
V. Surface Coatings and Devices incorporating Comb Copolymers
Numerous methods can be used to apply the comb copolymers, comb copolymer mixtures, or mixtures of comb copolymers and other polymers to surfaces. These methods include dip coating, spray coating, brush coating, roll coating, or spin casting a film onto the substrate followed by mild heating to promote adhesion to the surface. Solid free form processes such as 3DP, or freeze drying methods could be used to create complex three-dimensional structures, including porous structures. In all of these processing approaches a suitable crosslinking agent might be incorporated to enhance the mechanical rigidity of the coating or device.
In applications where mixtures of comb copolymers with other polymers are desirable, processing steps to achieve comb surface segregation include heating the mixture under vacuum, in air, water, water vapor, supercritical CO2 or other environment that favors the comb component at the surface, at temperatures sufficiently above the glass transitions of the polymers to provide the combs with the necessary mobility. In the case where the second polymer component is a semicrystalline polymer, the annealing temperature should be above the glass transition but below the melting point of the polymer, to ensure that the desired shape of the device is retained.
Surface segregation could be achieved preferably during a standard processing step in the manufacture of a biomedical device, such as during an extraction, autoclaving or sterilization process, or could be accomplished in a separate annealing step after the device has been manufactured. This type of processing creates a surface layer on the device that contains almost exclusively the comb copolymer. In other cases, the localization of the comb polymer to the surface of a device primarily comprised of a second polymer can be accomplished during other steps of device manufacture. For example, differences in viscosity between the comb copolymer and a second polymer when blended together can be exploited to locate the comb to the surface during melt extrusion of fibers, films or other devices. Porous or nonporous membranes, films, fibers or hollow fibers in which the comb copolymer resides at the surfaces can be prepared by phase inversion casting. In this method, a solution of the comb copolymer, the second polymer, and a mutual solvent is cast into an aqueous-based coagulation bath to form the device. During the casting process, favorable interactions between the comb and the coagulation bath medium induce segregation of the comb copolymer to exterior surfaces of the film, fiber, or membrane. Cell-regulating microporous biodegradable membranes useful as temporary barrier devices in wound-healing applications can be prepared in this fashion. Cell-regulating biodegradable sutures can similarly be prepared by spinning fibers from solution into an aqueous-based coagulation bath. Such surface-modified fibers can also be prepared from biodegradable or nonbiodegradable materials and fashioned into nonwoven fabric articles for biomedical applications including cell-regulating temporary barrier devices and biofiltration devices. Hollow nanoporous fibers can be prepared which have cell-regulating interior surfaces. By encapsulating cells in a portion of such a fiber, a long-term drug delivery implant could be prepared which secretes desirable products of cells in quantities regulated wholly or in part by tethered signals on the fiber inner surface. Cell-regulating biodegradable microporous scaffolds with a surface excess of comb copolymers can be prepared by freeze-drying methods by choosing a sublimating solvent which has preferential affinity to the comb copolymer component as compared to the second polymer component which forms the bulk of the device.
V. Biomedical Applications
The comb-type copolymers described herein may be used in a variety of biomedical applications, such as in scaffolds and supports for cell growth in tissue engineering, coatings for biomedical implants such as intraocular lenses or other permanent implants made from polymeric, metal, glass, or ceramic materials, and coatings for cell culture apparatus such as cell culture plates, pipets, etc.. The comb-type copolymers may be used for modifying the surface properties of sutures, temporary barrier films or fabrics in wound-healing applications, artificial hearts and blood vessels, catheters, filters for blood or other body fluids, and targeted controlled-release drug delivery vehicles and encapsulated cell drug delivery systems. The materials are preferably biodegradable when used for tissue engineering, wound healing, and targeted drug delivery applications, and are preferably non-degradable when used to modify implants, cell culture apparatus, filtration devices, and other devices intended for long term use or implantation.
A. Tissue Engineering
For use in tissue engineering applications, the comb copolymers may be derivatized by the attachment to the ends of the hydrophilic side chains biologically active molecules that promote favorable cell-polymer interactions, such as cell adhesion molecules and growth factors. Matrices suitable for seeding or ingrowth of cells can be formed which incorporate the comb copolymers, or a matrix formed of a material such as stainless steel, collagen, or another polymer can be coated with the comb copolymers. The matrix is then either seeded with cells and implanted, or the matrix implanted for tissue ingrowth to occur. These materials can be tailored to fit the particular needs of a variety of cell types through changes in the type and density of cell adhesion peptides attached to the copolymers. Cell types which can be seeded on the matrices include parenchymal cells such as hepatocytes, uroendothelial cells, skin cells, muscle cells, nerve cells and bone and/or cartilage forming cells. Normal cells, fetal cells or genetically engineered cells can be seeded onto the matrices.
B. Drug Delivery and Imaging
The comb copolymers also may be formed into matrices for use as drug delivery systems or for imaging purposes. Biodegradable latexes coated with the comb copolymers be can be used for targeted delivery of a therapeutic, prophylactic or diagnostic agent. Hollow nanoporous fibers can be prepared which have cell-regulating interior surfaces comprised of comb copolymers or comb copolymer mixtures. By encapsulating cells in a portion of such a fiber, a long-term drug delivery implant could be prepared which secretes desirable products of cells in quantities regulated wholly or in part by tethered signals on the fiber inner surface.
For use in drug delivery, a therapeutic or prophylactic agent, such as an amino acid, bioactive peptide or protein, carbohydrate, sugar, or polysaccharide, nucleic acid or polynucleic acid, synthetic organic compound, or metal may be attached to through the end groups of the hydrophilic side chains of the comb copolymer using methods available in the art. The comb copolymers may be modified to increase the level of the incorporated agent. Agents which provide greater stability for the agent to be delivered may be covalently or ionically attached to the copolymer. The comb copolymers may be functionalized with a specific binding moiety, e.g, an antibody, which targets the latex particle for delivery to a particular site within the body Hydrophilic, hydrophobic, acidic, basic or ionic side chains also may be attached to the copolymers to expand their use as delivery devices for drugs. Matrices of the modified drug-containing comb copolymer may be administered to an animal orally or parenterally to deliver the drug to the animal in vivo at a site in the animal where it is needed.
Diagnostic agents include radioactive materials, fluorescent materials, enzymatic materials, gases, and magnetic materials.
C. Use of the Materials to Provide Cell Repulsive Surfaces
It is often desirable to minimize cell and tissue interactions with biomedical implants, such as intraocular lenses. These interactions are minimized when the surface of an implant is coated with the non-cell binding copolymers. It is preferred that the copolymer be non-degradable in some applications. For example, when intraocular lenses are implanted, they are intended to remain in place for extended periods of time and biodegradability is to be avoided.
A preferred non-biodegradable polymeric material is a copolymer of an alkyl acrylate (i.e., methyl methacrylate) and PEG-methacrylate. A preferred method to place this coating at the surface is through the formation of a latex film.
The present invention will be further understood by references to the following non-limiting examples, in which the following materials and equipment were utilized.